S-adenosylmethionine Riboswitches Research Articles

Structure-based insights into recognition and regulation of SAM-sensing riboswitches.

Riboswitches are highly conserved RNA elements that located in the 5'-UTR of mRNAs, which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands. S-adenosylmethionine (SAM) is a ubiquitous methyl donor for transmethylation reactions in all living organisms. SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity, serving as regulatory modules in multiple metabolic pathways. To date, seven SAM-specific riboswitch classes that belong to four families, one SAM/SAH riboswitch and one SAH riboswitch have been identified. Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket. In this review, we summarize the current research progress on the distribution, structure, ligand recognition and gene regulation mechanism of these SAM-related riboswitch families, and further discuss their evolutionary prospects and potential applications.

Monitoring Single SAM/SAH Riboswitch Ligand-Induced Folding and Mimicking Co-Transcriptional Folding

Riboswitches are widely found in the 5’-UTR of prokaryotic genes, alternating downstream expression upon ligand-binding. The SAM (S-adenosyl methionine) riboswitches are located upstream of multiple genes related to methionine and cysteine biosynthesis. While most SAM riboswitches have strong discrimination between SAM and SAH, the SAM/SAH riboswitch shows almost identical binding affinity for both ligands. Previous NMR [1] and crystal structures [2] have revealed the ligand-binding conformation.

Cryo-EM structure of a 40\xa0kDa SAM-IV riboswitch RNA at 3.7\u2009\xc5 resolution

Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.

Energy landscape analysis for regulatory RNA finding using scalable distributed cyberinfrastructure

SUMMARYWe investigate the folding energy landscape for a given RNA sequence through Boltzmann ensemble (BE) sampling of RNA secondary structures. The ensemble of sampled structures is used to derive distributions of energies and base‐pair distances between two configurations. We identify structural features that can be utilized for RNA gene finding. Characterization of the EL through BE sampling of secondary structures is computationally demanding and has multiple heterogeneous stages. We develop the Distributed Adaptive Runtime Environment to effectively address the computational requirements. Distributed Adaptive Runtime Environment is built upon an extensible and interoperable pilot‐job and supports the concurrent execution of a broad range of task sizes across a range of infrastructure. It is used to investigate two RNA systems of different sizes, S‐adenosyl methionine (SAM) binding RNA sequences known as SAM‐I riboswitches, and the S gene of the bovine corona virus RNA genome. We demonstrate how the implementation lowers the total time to solution for increases in RNA length, the number of sequences investigated, and the number of sampled structures. The distributions of energies and base‐pair distances reveal variations in folding dynamics and pathways among the SAM riboswitch sequences. Our results for BCoV RNA genome sequences also indicate sensitivity of folding to coding‐neutral variations in sequence. We search for a characteristic motif from within the SAM‐I consensus structure – a four‐way junction, among BE sampled structures for all 2910 SAM‐I sequences identified from Rfam (the curated ncRNA family database). We find that BE sampling provides insight into the variations in conformational distribution among sequences of the same ncRNA family. Therefore, BE sampling of secondary structures is a viable pre‐processing or post‐processing tool to complement comparative sequence analysis. The understanding gained shows how appropriately designed cyberinfrastructure can provide new insight into RNA folding and structure formation. Copyright © 2011 John Wiley & Sons, Ltd.

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

A trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria monocytogenes

Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5'-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.

Folding of the SAM Aptamer is Determined by the Formation of a K-turn-dependent Pseudoknot

The S-adenosylmethionine (SAM) riboswitch is one of the most recurrent riboswitches found in bacteria and has three known different natural aptamers. The Bacillus subtilis yitJ SAM riboswitch aptamer is organized around a four-way junction which is characterized by the presence of a pseudoknot and a K-turn motif. By replacing the adenine involved in a Watson-Crick base pair at position 138 in the core region of the aptamer with the fluorescent analogue 2-aminopurine (2AP), we show that the ligand-induced reorganization of the aptamer strongly attenuates 2AP fluorescence. The fluorescence quenching process is specific to SAM on the basis of the observation that the structural analogue S-adenosylhomocysteine does not promote a similar effect. We find that the pseudoknot is important for the reorganization of the core domain and that the K-turn motif also has a marked influence on the core domain reorganization, most probably through its important role in pseudoknot formation. Finally, we show that SAM riboswitch ligand binding is facilitated by the L7Ae K-turn binding protein, which suggests that K-turn motifs may be protein anchor sites used by riboswitches to promote RNA folding.

Structural Studies of the Purine and SAM Binding Riboswitches

Riboswitches are recently discovered genetic regulatory elements found in the 5'-untranslated regions of bacterial mRNAs that act through their ability to specifically bind small-molecule metabolites. Binding of the ligand to the aptamer domain of the riboswitch is communicated to a second domain, the expression platform, which directs transcription or translation of the mRNA. To understand this process on a molecular level, structures of three of these riboswitches bound to their cognate ligands have been solved by X-ray crystallography: the purine, thiamine pyrophosphate (TPP), and S-adenosylmethionine (SAM-I) binding aptamer domains. These studies have uncovered three common themes between the otherwise different molecules. First, the natural RNA aptamers recognize directly or indirectly almost every feature of their ligand to achieve extraordinary specificity. Second, all of these RNAs use a complex tertiary architecture to establish the binding pocket. Finally, in each case, ligand binding serves to stabilize a helix that communicates the binding event to the expression platform. Here, we discuss these properties of riboswitches in the context of the purine and SAM-I riboswitches.

Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria

Comparative sequence analysis and structural probing identified five RNA elements in the intergenic region of Agrobacterium tumefaciens and other α-proteobacteria. One of these RNA elements is probably a SAM-II, the only riboswitch class identified so far that is not found in Gram-positive bacteria.

An mRNA structure that controls gene expression by binding S-adenosylmethionine.

Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs. These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite. Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity. SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis. This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.